Functionalized fluorescent nanocrystal compositions and methods of making

ABSTRACT

The present invention provides for functionalized fluorescent nanocrystal compositions and methods for making these compositions. The compositions are fluorescent nanocrystals coated with at least one material. The coating material has chemical compounds or ligands with functional groups or moieties with conjugated electrons and moieties for imparting solubility to coated fluorescent nanocrystals in aqueous solutions. The coating material provides for functionalized fluorescent nanocrystal compositions which are water soluble, chemically stable, and emit light with a high quantum yield and/or luminescence efficiency when excited with light. The coating material may also have chemical compounds or ligands with moieties for bonding to target molecules and cells as well as moieties for cross-linking the coating. In the presence of reagents suitable for reacting to form capping layers, the compounds in the coating may form a capping layer on the fluorescent nanocrystal with the coating compounds operably bonded to the capping layer.

CROSS REFERENCES AND RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. Utility patentapplication Ser. No. 10/245,082, filed Sep. 17, 2002 which claimspriority from U.S. Provisional Application Ser. No. 60/379,208, filedMay 9, 2002, and U.S. Provisional Application Ser. No. 60/322,982, filedSep. 17, 2001, now U.S. patent application Ser. No. 10/245,082, thedisclosures of which are incorporated herein by reference in theirentirety; this application is also related to application Ser. No.______ [not yet assigned] filed on the same date as this application,entitled “HIGHLY LUMINESCENT FUNCTIONALIZED SEMICONDUCTOR NANOCRYSTALSFOR BIOLOGICAL AND PHYSICAL APPLICATIONS”, Attorney Docket No,126433.1101, the contents of which is herein incorporated by reference.The incorporation by reference of these documents is not admitted to beprior art with respect to the present invention by its mention in theCross Reference.

GOVERNMENT INTEREST

[0002] This invention was made, in part, with government support undercontract DAAD17-01-C-0024 with the United States Army ResearchLaboratory. The government has certain rights in this invention.

FIELD OF INVENTION

[0003] This invention relates to surface modification of nanocrystalswith a special chemistry that imparts to the semiconductor nanocrystalsproperties, which include water solubility, protection from dissolution,protection from photo-auto-oxidation, functionalization, and a markedenhancement of fluorescence intensity.

BACKGROUND OF THE INVENTION

[0004] Fluorescence-based analysis has become a powerful tool inscientific research, clinical diagnostics and many industrialapplications. However, while fluorescent organic molecules such asfluorescein and phycoerethrin are used frequently in fluorescencedetection systems, there are disadvantages in using these moleculesseparately or in combination. For example, photobleaching (fading ofintensity under light sources) is a major problem that hinders theaccuracy of quantitative measurements made using these molecules. Inaddition, each type of fluorescent molecules typically requiresexcitation with photons of a different wavelength as compared to thatrequired for another type of fluorescent molecules due to the relativelynarrow absorption spectrum of each. Moreover, even when a single lightsource is used to provide a single excitation wavelength, often there isoverlapping or insufficient spectral spacing between the emissions ofdifferent fluorescent molecules to permit individual and quantitativedetection.

[0005] Semiconductor nanocrystals are now being evaluated as a promisingtool of nonisotopic detection to replace conventional fluorescentmolecules. Since the spectral emission characteristics of nanocrystalsare a function of the size, nanocrystals produced in a narrow sizedistribution can be excited to emit a discrete fluorescence peak ofnarrow bandwidth. In other words, the ability to control the spectralcharacteristics of nanocrystals (narrow bandwidth, discrete emissionwavelengths, a single wavelength can excite an array of nanocrystalswith different emissions) is the major attracting point in their use.Another advantage of the nanocrystals is their resistance towardphotobleaching under intensive light sources.

[0006] Examples of semiconductor nanocrystals are known in the art tohave a core selected from the group consisting of CdSe, CdS, CdTe(collectively referred to as “CdX”) (see, e.g., Norris et al., 1996,Physical Review B. 53: 16338-16346; Nirmal et al., 1996, Nature 383:802-804; Empedocles et al., 1996, Physical Review Letters 77: 3873-3876;Murray et al., 1996, Science 270: 1355-1338; Effros et al., 1996,Physical Review B. 54: 4843-4856; Sacra et al., 1996, J. Chem. Phys.103: 5236-5245; Murakoshi et al., 1998, J. Colloid Interface Sci. 203:225-228; Optical Materials and Engineering News, 1995, Vol. 5, No. 12;and Murray et al., 1993, J. Am. Chem. Soc. 115: 8706-8714; thedisclosures of which are hereby incorporated by reference), and ZnS (Khoet al. 2000, Biochem. Biophys. Research Commun. 272: 29-35).

[0007] As known in the art, a manual batch method may be used to preparesemiconductor nanocrystals of relative monodispersity (e.g., thediameter of the core varying approximately 10% between quantum dots inthe preparation), as has been described previously (Bawendi et al.,1993, J. Am. Chem. Soc. 115: 8706). Advances in nanocrystal coreproduction and improvements in narrowing the particle size distribution,the controllability of particle size, and the reproducibility ofproduction have been achieved by a continuous flow process (U.S. Pat.No. 6,179,912, the disclosure of which is herein incorporated byreference). Core semiconductor nanocrystals, however, exhibit lowfluorescence intensity upon excitation, lack of water solubility, lackof surface functional groups for linking with target molecules, andadditionally, susceptibility to dissociation and degradation in aqueousenvironments with high ionic strength. The low fluorescence intensityhas been ascribed to a leak of exciton to the outer phase, to thepresence of surface energy states that act as traps which degrade thefluorescence properties of the core nanocrystal, or to intermediatestates of non radiative relaxations.

[0008] Efforts to improve the fluorescence intensity involve passivating(or capping) the outer surface of a core nanocrystal in order to reduceor eliminate the surface energy states. Inorganic materials with higherband gap energy have been used for passivation; i.e., core nanocrystalshave been passivated with an inorganic coating (“shell”) uniformlydeposited on the surface of the core nanocrystals. The shell which istypically used to passivate CdX core nanocrystals is preferablycomprised of YZ wherein Y is Cd, Hg, or Zn, and Z is S, or Se, or Te.However, these passivated semiconductor nanocrystals have been reportedto have a limited improvement in fluorescence intensity (with referenceto quantum yield), and to have solubility in organic solvents only.Organic molecules, such as tri-n-octyl phosphine (TOP) and tri-n-octylphosphine oxide (TOPO) have been also used to coat nanocrystals (seeMurray et al., 1993, J. Am. Chem. Soc. 115: 8706-8714, Hines andGuyot-Sionnest 1996, J. Phys. Chem. 100: pp 468, Dabbousi et al., 1997,J. Phys. Chem. 101: 9463). Coatings with these molecules have beenreported to produce limited improvement in fluorescence intensity, andprovide the coated nanocrystals with solubility only in organicsolvents. Furthermore, the molecules coating the nanocrystal are easilydisplaced by different solvents.

[0009] To make fluorescent nanocrystals useful in biologicalapplications or detection systems utilizing an aqueous environment, itis desirable that the fluorescent nanocrystals used in the detectionsystem are water-soluble. “Water-soluble” is used herein to meansufficiently soluble or suspendable in an aqueous solution, such as inwater or water-based solutions or buffer solutions, including those usedin biological or molecular detection systems as known by those skilledin the art. Particles and surfaces may also be characterized by theirability to be wet by a fluid. The fluid may be water or a solution ofwater and other liquids like ethanol. One method to impartwater-solubility or wettability to semiconductor nanocrystals (e.g., CdXcore/YZ shell nanocrystals) is to exchange the overcoating layer of TOPor TOPO with a coating, or “capping compound”, which will impart somewater-solubility. For example, a mercaptocarboxylic acid may be used asa capping compound to exchange with the organic layer (see, e.g., U.S.Pat. No. 6,114,038, the disclosure of which is herein incorporated byreference; see also, Chan and Nie, 1998, Science 281: 2016-2018). Thethiol group of monothiol capping compound bonds with the Cd—S or Zn—Sbonds (depending on the composition of the nanocrystal), creating acoating which is to some extent not easily displaced in solution, andimparting some stability to the nanocrystals in suspension.

[0010] Another method to make the CdX core/YZ shell nanocrystalswater-soluble or wettable is by the formation of a coating of silicaaround the semiconductor nanocrystals (Bruchez, Jr. et., 1998, Science281: 2013-2015; U.S. Pat. No. 5,990,479) utilizing a mercapto-basedlinker to link the glass to the semiconductor nanocrystals. Anextensively polymerized polysilane shell has been reported to impartwater solubility or wettability to nanocrystalline materials, as well asallowing further chemical modifications of the silica surface.

[0011] Depending on the nature of the coating compound, coatedsemiconductor nanocrystals which have been reported as water-soluble mayhave limited stability in an aqueous solution, particularly when exposedto air (oxygen) and/or light. For example, oxygen and light can causemercapto-based monothiols used in capping and passivation ofnanocrystals to become catalytically oxidized, thereby formingdisulfides which destabilize the attachment of the coating and mighteven play a role in oxidizing the core semiconductor (see, e.g., Aldanaet al., 2001, J. Am. Chem. Soc. 123: 8844-8850). Thus, oxidation maycause the capping layer to migrate away from the surface of thenanocrystals, thereby exposing the surface of the nanocrystals resultingin “destabilized nanocrystals” that eventually form nonsolubuleaggregates with low fluorescence intensity. In addition, current meansfor passivating semiconductor nanocrystals are still rather inefficientin increasing the fluorescence intensity to a level desired fordetection systems (e.g., in providing a significant increase insensitivity in fluorescence-based detection systems as compared tocurrently available fluorescent dyes).

[0012] As is evident from current progress in the process of producingsemiconductor nanocrystals, it is important to supply the nanocrystalswith a stable, and protective capping layer to achieve the desiredcombinations of properties. In other words, it is desirable that thecapping layer be designed in such a way that it is able to impart to thesemiconductor nanocrystals improvement in fluorescence efficiency(quantum yield); water solubility; stability in aqueous solutions;stability in media with high ionic strength; resistance to the exposureto hostile environment with light, oxygen and ions; and the ability tobind ligands, molecules, probes of various types, and solid supports.Additionally, there remains a need for a nonisotopic detection systemwhich results in generation of a signal comprising fluorescence emissionof high intensity; can result in signal amplification; is not limited asto the chemical nature of the target molecule to be detected (e.g.,versus detection of nucleic acid molecules only); can be used to bindmolecular probes of various types (affinity molecules, oligonucleotides,nucleobases, and the like); and can result in simultaneous detection ofmore than one type of target molecule by utilizing a class ofnonisotopic molecules that may be excited with a single excitation lightsource and with resultant fluorescence emissions with discretefluorescence peaks that can be spectrally distinguished from each other(e.g., using detection means for fluorescence that is standard in theart).

[0013] It is an object of the present invention to provide fluorescentnanocrystals which provides a combination of properties including asignificant enhancement of fluorescence intensity, water solubility,physical and chemical stability, and functionalization.

SUMMARY OF THE INVENTION

[0014] The present invention provides for functionalized fluorescentnanocrystal compositions and methods for making these compositions. Thecompositions are fluorescent nanocrystals coated with at least onematerial. The coating material has chemical compounds or ligands withfunctional groups or moieties with conjugated electrons and moieties forimparting high luminescence efficiency and solubility to coatedfluorescent nanocrystals in aqueous solutions. The coating materialprovides for functionalized fluorescent nanocrystal compositions whichare water soluble, chemically stable, and emit light with a quantumyield of greater than 10% and preferably greater than 50% when excitedwith light. The coating material may also have chemical compounds orligands with moieties for bonding to target molecules and cells as wellas moieties for cross-linking the coating. In the presence of reagentssuitable for reacting to form capping layers, the compounds in thecoating may form a capping layer on the fluorescent nanocrystal with thecoating compounds operably bonded to the capping layer.

[0015] The present invention provides compositions comprisingfunctionalized, stable, and fluorescent nanocrystals for use innonisotopic detection systems and methods of making the same. Thefunctionalized fluorescent nanocrystals are coated with a materialcomprised of a heteroaromatic compound or ligand with functional groupsor moieties for imparting solubility to coated fluorescent nanocrystalsin aqueous solutions. The coating material provides for functionalizedfluorescent nanocrystal compositions which are water soluble, chemicallystable, and emit with high efficiency light with a quantum yield ofgreater 10% and preferably greater than 50% when excited with light.Depending upon the ligands comprising the material coating thefluorescent nanocrystals, the functionalized fluorescent nanocrystals ofthe present invention may be soluble in other liquids, for example waterand isopropyl alcohol mixture or liquids with surface tensions belowabout 80 dynes/cm, and preferably in the range from about 30-72dynes/cm. The coating material may also have chemical compounds orligands like isocyanates, alkyl cyanoacrylates, or alkyl phosphines withmoieties for bonding to target molecules and cells as well as moietiesfor cross-linking the coating. In the presence of suitable reagents forexample but not limited to ZnSO₄ and Na₂S, the compounds in the coatingmay form a capping layer on the fluorescent nanocrystal with the coatingcompounds operably bonded to the capping layer.

[0016] The present invention provides compositions comprisingfunctionalized, stable, and fluorescent nanocrystals for use innonisotopic detection systems and methods of making the same. Thefluorescent nanocrystal composition is a core fluorescent nanocrystalcoated with an imidazole-containing compound cross-linked with an alkylphosphine-containing compound and complexed (e.g., by adduct formation)with an inorganic semiconductor capping layer.

[0017] The coating materials of the present invention enhance thefluorescence intensity of the core nanocrystal by several folds.Further, the functionalized, fluorescent nanocrystals according to thepresent invention are very soluble and stable in aqueous media atdifferent pH levels.

[0018] The functionalized nanocrystals described in this inventiondisplay an unexpected increase in fluorescence intensity as compared toother semiconductor nanocrystal formulations known in the art.Intra-unit energy transfer between the imidazoles and the corenanocrystals may be playing a major role in the fluorescenceenhancement. Other mechanisms may also account for such increase influorescence intensity imparted to these functionalized, fluorescentnanocrystals, may essentially include, but are not limited to,eliminating mid-band and intermediate states, passivation, chargedislocation, higher band energy condensation and resonance, modificationof the bandgap of the shell semiconductor, or a combination thereof. Theconcept of passivation has been previously described herein. Thepassivating effect is due to the capping of surface Cd or Zn atoms orthe like by imidazole complexation and to the capping of the counteratoms (Se or S or the like) by complexation with the alkylphosphine-containing compounds. As to charge dislocation, imidazole andalkyl phosphine moieties present in the coating, may be susceptible toexcitation by a sufficient light source. Such excitation may lead tocharge transfer from the imidazole and/or alkyl phosphine moieties tothe nanocrystal structure thereby resulting in an increase influorescence intensity, as compared to a fluorescence intensity withoutsuch internal charge transfer (internal charge transfer meaning atransfer of energy that substantially comprises a transfer within thestructure of a coated nanocrystal of which the imidazole and/or alkylphosphine are part of, as opposed to a transfer limited only to atransfer of energy between neighboring nanocrystals). As for energycondensation and fluorescence, it is anticipated that the integration ofthe π electrons in the imidazole and phosphine moieties with the excitedelectrons at the higher energy band of the core crystal is producing anenergy condensation effect that yields higher level of electron-holecoupling followed by radiative relaxation. Chemical modifications ofsemiconductor surfaces with organic or inorganic molecules can shift theband edges of a semiconductor positive or negative (JECS 142, 2625,(1995)). Since strong surface capping and passivation of coresemiconductor nanocrystals by higher band gap energy materials ororganic passivators (e.g., TOPO) do not significantly enhance thefluorescence intensity of core semiconductor nanocrystals, it seems thatenergy condensation, energy transfer and eliminating intermediate andmid band exciton states are the major factors that underlie thefluorescence enhancement. The functionalized, fluorescent nanocrystalsof the present invention are functionalized to be water-soluble and tocontain one or more reactive functionalities to which a molecular probemay be operably bound. The overall structure of the functionalized,fluorescent nanocrystals of the present invention closely resembles thestructure of a miniature light emitting diode (LED) where the coatingmaterials used in this invention play the roles of hole-blocking layerand electron-transport layer.

[0019] Luminescence is the emission of light from any substance andoccurs from electronically excited states. Luminescence is divided intotwo categories, fluorescence and phosphorescence, depending on thenature of the excited state. When the return of the excited electrons tothe ground state is spin-allowed, the return is fast and this process istermed fluorescence (typically, a 10 nano second process). When thereturn of electrons to the ground states is spin forbidden it takeslonger time (>100 ns), and the emitted light is termed phosphorescence.Luminescence efficiency means the amount of light (in candela units forexample) that can be produced from a certain amount of nanocrystalsusing unlimited source of energy, for example beta emitter, light,recombination of injected charge carriers, or electrical current. Theterm quantum yield or efficiency in this case is different because itdescribes the ratio of energy recovery after applying certain amount ofenergy as photons, while, luminescence efficiency is different becauseit describes the full capacity of the nanocrystals (or a light source)apart from the amount of energy applied. In the practice of embodimentsof the present invention either the Quantum yield the luminescenceefficiency or both are increased by the coating material compositions ofthis invention.

[0020] In other embodiments of this invention, the ligands offunctionalized fluorescent nanocrystal may increase the fluorescenceefficiency (quantum yield) upon binding to the surface of thenanocrystal. In another embodiment of this invention, the ligands of thefunctionalized fluorescent nanocrystals may increase the fluorescenceefficiency by one or more of the following mechanisms: internal energytransfer (antenna effect), energy condensation, elimination ofintermediate and mid band exciton states, and surface passivation.

[0021] The above and other objects, features, and advantages of thepresent invention will be apparent in the following DETAILED DESCRIPTIONof the invention when read in conjugation with accompanying drawings inwhich reference numerals denote the same or similar parts throughout theseveral illustrated views and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1A is a schematic representation of the new coating systemand a process for its formation. FIG. 1B is the anticipated energychange diagrams during the coating process according to the presentinvention. FIG. 1C is an illustration of the embodiment of the presentinvention.

[0023]FIG. 2 is a graph showing a comparison of emission peaks offunctionalized fluorescent nanocrystals of the present invention andmercapto-based functionalized nanocrystals.

[0024]FIG. 3A comparison of UV and fluorescence spectra and quantumyield for fluorescein isothiocyanate (FITC) and functionalizedfluorescent nanocrystals of an embodiment of the present invention.Quantum yield (QY) value of the functionalized nanocrystals wasdetermined relative to FITC (QY=0.75˜0.95) in sodium borate buffer (50mM, pH 9.0) using the equation: QYNC=QYFITC(INC/IFITC) (AFITC/ANC)(n/n′)2, where, INC and IFITC are the integrated fluorescence intensityof nanocrystals and FITC, respectively; and ANC and AFITC are the UVabsorbance maxima at excitation for nanocrystals and FITC respectively;and n and n′ are the refractive indices of the solvents (sodium boratefor both samples). According to the above equation the QYNC=0.73˜1.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Throughout the specification of the application, various termsare used such as “primary”, “secondary”, “first”, “second”, and thelike. These terms are words of convenience in order to distinguishbetween different elements, and such terms are not intended to belimiting as to how the different elements may be utilized.

[0026] By the term “ target molecule” is meant, for the purpose of thespecification and claims to refer to a molecule of an organic orinorganic nature, the presence and/or quantity of which is being testedfor; and which contains a molecular component (e.g., ligand or sequenceor epitope or domain or portion or chemical group or reactivefunctionality or determinant, or the like) for which a molecular probehas binding specificity. The molecule may include, but is not limitedto, a nucleic acid molecule, protein, glycoprotein, eukaryotic cell,prokaryotic cell, lipoprotein, peptide, carbohydrate, lipid,phospholipid, aminoglycans, chemical messenger, biological receptor,structural component, metabolic product, enzyme, antigen, drug,therapeutic, toxin, inorganic chemical, organic chemical, a substrate,and the like. The target molecule may be in vivo, in vitro, in situ, orex vivo.

[0027] By the term “molecular probe” is meant, for purposes of thespecification and claims, to mean a molecule which has bindingspecificity and avidity for a molecular component of, or associatedwith, a target molecule. In general, molecular probes are known to thoseskilled in the art to include, but are not limited to, lectins orfragments (or derivatives) thereof which retain binding bindingfunction, monoclonal antibodies (“mAb”, including chimeric orgenetically modified monoclonal antibodies which may be preferable foradministration to humans). Peptides, aptamers, and nucleic acidmolecules (including but not limited to, single stranded RNA orsingle-stranded DNA, or single-stranded nucleic acid hybrids,oligonucleotide analogs, backbone modified oligonucleotide analogs,morpholino-based polymers), and nucleobases. The term “nucleobase” isused herein to refer to a nucleic acid moiety including, but not limitedto: nucleosides (including derivatives, or functional equivalentsthereof, and synthetic or modified nucleosides, and particularly, anucleoside comprising a reactive functionality (e.g., free amino groupor carboxyl group)); nucleotides (including dNTPs, ddNTPs, derivativesor functional equivalents thereof, and particularly, a nucleotidecomprising a reactive functionality (e.g., free amino group or carboxylgroup); acyclonucleoside triphosphates (see, e.g., U.S. Pat. No.5,558,991); 3′(2′)-amino-modified nucleosides, 3′(2′)-amino-modifiednucleotides, 3′(2′)-thiol-modified nucleosides, 3′(2′)-thiol-modifiednucleotides (see, e.g., U.S. Pat. No. 5,679,785); alkylamino-nucleotides(see, e.g., as a chain terminator, U.S. Pat. No. 5,151,507); nucleosidethiotriphosphates (see, e.g., U.S. Pat. No. 5,187,085); and the like.The term “monoclonal antibody” is also used herein, for purposes of thespecification and claims, to include immunoreactive fragments orderivatives derived from a mAb molecule, which fragments or derivativesretain all or a portion of the binding function of the whole mAbmolecule. Such immunoreactive fragments or derivatives are known tothose skilled in the art to include F(ab′)2, Fab′, Fab, Fv, scFV, Fd′and Fd fragments. Methods for producing the various fragments orderivatives from mABs are well known in the art (see, e.g., Pluckthum,1992, Immunol. Rev. 130:152-188; for example, via pepsin digestion,papain digestion, reduction of disulfide bridges, and methods describedin U.S. Pat. No. 4,6142,334). Single chain antibodies can be produced asdescribed in U.S. Pat. No. 4,946,778. The construction of chimericantibodies is now a straightforward procedure (Adair, 1992,Immunological Reviews 130: 5-40) in which the chimeric antibody is madeby joining the murine variable region to a human constant region.Additionally, “humanized” antibodies may be made by joining thehypervariable regions of the murine monoclonal antibody to a constantregion and portions of variable region (light chain and heavy chain)sequences of human immunoglobulins using one of several techniques knownin the art (Adair, 1992, supra; Singer et al., 1993, J. Immunol. 150:2844-2857). Methods for making a chimeric non-human/human mAb in generalare described in detail in U.S. Pat. No. 5,736,137. Aptamers can be madeusing methods described in U.S. Pat. No. 5,789,157. Lectins andfragments thereof are commercially available. Oligonucleotide analogs,backbone modified oligonucleotide analogs, and morpholino-based polymerscan be made using methods described in U.S. Pat. Nos. 5,969,135, and 5,596, 086, U.S. Pat. Nos. 5,602,240, and 5, 034, 506, respectively.“Molecular probe” may also be used herein to refer to a plurality ofmolecules of molecular probe which may be operably bound to afunctionalized, fluorescent nanocrystal.

[0028] By the terms “operably bind” and “operably bound” are meant, forpurposes of the specification and claims to refer to fusion or bond oran association, of sufficient stability for the purposes of use indetection systems as described herein and standard conditions associatedtherewith as known in the art, formed between a combination of differentmolecules including, but not limited to, between a coating compound andfluorescents nanocrystal, between a coating compound and molecularprobe, between different molecular probes, and between molecular probeand target molecule. A coating may comprise one or more ligands. Asknown to those skilled in the art, and as will be more apparent by thefollowing embodiments, there are several methods and compositions inwhich two or more molecules may be operably bound utilizing reactivefunctionalities. Reactive functionalities include, but are not limitedto, bifunctional reagents (e.g., homobifunctional orheterobifunctional), biotin, avidin, free chemical groups (e.g., thiol,or carboxyl, hydroxyl, amino, amine, sulfo, and the like), and reactivechemical groups (reactive with free chemical groups). As known to thoseskilled in the art, the bond may compromise, but is not limited to, oneor more of: covalent, ionic, hydrogen, van der waals, and the like.

[0029] By the term “imidazole-containing compound” is meant, forpurposes of the specification and claims to refer to a heterocyclic orheteroaromatic molecule or ligand in a coating that has at least oneimidazole group (e.g., imidazole ring) available for binding with thefluorescent nanocrystal or capping compound a metal such as zinc,cadmium, gallium, or other metal cation, or substrate containing suchcation. In that respect, preferably at least one imidazole moiety is ina terminal position with respect to the structure of the molecule. Theimidazole containing compound operably bonds to the fluorescentnanocrystal through the imidazole ring which comprises delocalizedmolecular orbitals. Generally, imidazole ring nitrogens serve ascoordinating ligand, as illustrated in FIG. 1, to operably bind a metalion such as zinc or cadmium. In a preferred embodiment, theimidazole-containing compound comprises additional reactivefunctionalities such as an amino acid, or two or more amino acids joinedtogether (e.g., known in the art as “peptidyl” or “oligopeptide”), whichmay include, but is not limited to, histidine, carnosine, anserine,baleine, homocarnosine, histidylphenylalanine,cyclo-histidylphenylalanine, 5-amino-4-imidazolecarboxamide,histidylleucine, 2-mercaptoimidazole, boc-histidine hydrazide,histidinol, 1-methylhistidine, 3-methylhistidine, imidazolysine,imidazole-containing ornithine (e.g., 5-methylimidazolone),imidazole-containing alanine (e.g., (beta)-(2-imidazolyl)-L(alpha)alanine), carcinine, histamine, and the like. These histidine-basedmolecules or imidazole-containing amino acids, may be synthesized usingmethods known in the art (see, e.g., Stankova et al., 1999, J. PeptideSci. 5: 392-398, the disclosure of which is herein incorporated byreference).

[0030] By the term “amino acid” is meant, as known in the art and forpurposes of the specification and claims, to refer to a compound orligand containing at least one amino group and at least one carboxylgroup. As known in the art, an amino group may occur at the positionadjacent to a carboxyl group, or may occur at any location, for exampleβ and γ amino acids, along the amino acid molecule. In addition to atleast one imidazole moiety, the amino acid may further comprise one ormore additional reactive functionalities (e.g., amino, thiol, carboxyl,carboxamide, etc.). The amino acid may be a naturally occurring aminoacid, a synthetic amino acid, a modified amino acid, an amino acidderivative, an amino acid precursor, in D (dextro) form, or in L (levo)form. Examples of derivatives may include, but are not limited to, ann-methylated derivative, amide, or ester, as known in the art.Consistent with the functionality of the amino acid, it acts as acoating for the fluorescent nanocrystals and may impartwater-solubility, buffer sufficiently in a pH range between about pH 6and about pH 10, functions as a coat which can increase fluorescenceintensity, and has one or more reactive functionalities that may be usedto operably bind at least one molecular probe. An amino acid of theaforementioned amino acids may be used in a preferred embodiment, and apreferred amino acid may be used separately in the composition of thepresent invention to the exclusion of amino acids other than thepreferred amino acid. Carnosine (alanyl histidine) is a preferredhistidine-based or imidazole-containing compound for coating thefunctionalized, fluorescent nanocrystals according to the presentinvention.

[0031] Other molecules or ligands may be used in place of imidazole inthe practice of this invention. These ligands, (such as those disclosedin Cotton and Wilkinson, 3^(rd) Ed, Chapter 21) may operably bond tofluorescent nanocrystal surfaces, coordinate or chelate metal ions andpreferably have Lewis base properties and or conjugated moieties. Thesemolecules may also have moieties for imparting solubility or wettabilityin aqueous solutions to fluorescent nanocrystals coated with them. Themolecules may also have chemical moieties for bonding to targetmolecules and cells as well as moieties for cross-linking them. In thepresence of reagents suitable reagents like ZnSO₄ and Na₂S, thesemolecules or compounds may react to form a capping layer on thefluorescent nanocrystal with the molecules operably bonded to thecapping layer. The molecules may also operably bond to atoms or ions onthe surface of the nanocrystal. The result of using these molecules orcompounds is a functionalized nanocrystal with enhanced luminescence,water solubility and chemical stability. These molecules or compoundshave at least one Lewis acid and or a conjugated moiety in a terminalposition with respect to the structure of the molecule. Generally,cyclic or linear unsaturated compounds with resonating electrons or withresonating heterogeneous rings frequently serve as coordinating ligandto operably bind a metal ion such as zinc or mercury, or cadmium. Theligand bonds to the fluorescent nanocrystal with its moiety thatcomprises delocalized molecular orbitals as illustrated in FIG. 1. Thesemolecules or compounds may comprise a heterogeneous ring, or two or morejoined together, examples include, but are not limited to, thiazole,thiazole derivatives, oxazole, oxazole derivatives, pyrrole, pyrrolederivatives including doped or undoped poly-pyrrole oligomers,thiophene, thiophene derivatives including doped or undopedpoly-thiophenes, furan, furan derivatives, pyridine, pyridinederivatives, pyrimidine, pyrimidine derivatives, pyrazine, pyrazinederivatives, triazine, triazine derivatives, triazole, triazolederivatives, phthalocyanine, phthalocyanine derivatives, porphyrin,porphyrin derivatives. These compounds may comprise unsaturated (olefin)hydrocarbons or their amine, phosphorus, oxygen derivatives, which mayinclude but are not limited to acetylene, propyne, and allene. It ispreferred that the molecule have suffucient p-electron density to engagein adduct formation or resonance on the surface of the semiconductornanocrystal.

[0032] By the term “alkyl phosphine cross-linking compound” is meant,for purposes of the specification and claims to refer to a molecule orligand that has at least one phosphine group available for binding orchelating a non metal such as Se, S or other non metals, or substratecontaining such atoms, and has at least one functional group (e.g.,hydroxyl, amino, thiol, carboxyl, carboxamide, etc) with ability toreact with neighboring molecules. In that respect, preferably at leastone phosphine moiety is in a terminal position with respect to thestructure of the molecule as illustrated in FIG. 1. Generally, phosphinemoieties frequently serve as coordinating ligand to operably bind withthe fluorescent nanocrystal or capping compound a non metal or ion suchas Se or S. In a preferred embodiment, the alkyl phosphine-containingcompound comprises a phosphine group, or two or more phosphine groupsjoined together (e.g., in a polymeric form), which may include, but isnot limited to, hydroxymethylphosphine compounds, and the like. Alkylphosphine-containing compounds may be synthesized using methods known inthe art (see, e.g., Tsiavaliaris et al., 2001, Synlett. 3: 391-393,Hoffman et al, 2001, Bioconjug Chem 12: 354-363, U.S. Pat. No.5,948,386). As known in the art, an alkyl phosphine-containing compoundmay further comprise one or more additional reactive functionalities(e.g., hydroxyl, amino, thiol, carboxyl, carboxamide, etc.). Examples ofderivatives may include, but are not limited to, a hydroxy methylphosphine derivative, amide, or ester, as known in the art, and whereconsistent with the functions of the alkyl phosphine as a coating asdescribed herein (e.g., imparts water-solubility, buffers sufficientlyin a pH range between about pH 6 and about pH 10, functions as a coatand cross-linker which can increase stability and fluorescenceintensity, and has one or more reactive functionalities that may be usedto operably bind molecular probe). An alkyl phosphine of theaforementioned derivatives may be used in a preferred embodiment, and apreferred alkylphosphine may be used separately in the composition ofthe present invention to the exclusion of alkyl phosphines other thanthe preferred alkyl phosphine. Tris (hydroxy methyl) phosphine andbeta-[Tris(hydroxymethyl)phosphino]propionic acid are particularlypreferred alkyl phosphine-containing compound for coating, stabilizingand functionalizing fluorescent nanocrystals according to the presentinvention. Also known in the art is that cross-linked alkylphosphine-containing compounds have additional ability to operably bindto metal atoms and/or ions such as zinc and cadmium. In this respectfunctionalized isocyanates or alkyl cyanoacrylates may also be usefulfor cross-linking ligands and adduct formation with fluorescentnanocrystals in the practice of this invention.

[0033] The nanocrystal coated with the coating material of the presentinvention may further comprise an additional layer on the surface of thecoating material. The moieties of the layer may be organic or inorganicand provide chemical compatibility, reactivity, or solubility with afluid or suspension media. For example, additional amino acids likearginine may be coupled to the imidazole-containing group in the coatingmaterial, or short chain polymer or peptide sequences likearginine-glycine-serine, with the serine hydroxyl moiety interactingwith the suspending medium, may be used in the practice if thisinvention. Amino acids and other such groups will have an affinity forthe amino acid portion of the imidazole containing group of the coatingmaterial and may be reacted with them using standard coupling reactions.

[0034] By the term “fluorescent nanocrystals” is meant, for purposes ofthe specification and claims to refer to nano-crystals comprisingsemiconductor nanocrystals or doped metal oxide nanocrystals, to whichmay be operably bound various ligands including histidine-based orimidazole-containing compounds and phosphonium compounds. “Semiconductornanocrystals” is meant, for purposes of the specification and claims torefer to quantum dots (also known as crystallite semiconductors)comprised of a core comprised of at least one of a Group II-VIsemiconductor material (of which ZnS, HgS, and CdSe are illustrativeexamples), or a Group III-V semiconductor material (of which GaAs is anillustrative example), or a Group IV semiconductor nanocrystal, or acombination thereof. These core semiconductor nanocrystals may furthercomprise and be passivated with a “shell” or capping layer of materialuniformly deposited on the core. The capping layer material may becomprised of an inorganic material with a higher band gap than the corenanocrystal. Inorganic materials typically used to passivate CdX (X═S,Se, Te) core nanocrystals are preferably comprised of YZ where “Y” isCd, Hg, or Zn and “Z” is S, Se, or Te. Core CdX nanocrystals with a YZshell can be made by the methods in: (see, e.g., Danek et al., 1996,Chem. Mater. 8: 173-179; Dabbousi et al., 1997, J. Phys. Chem. B 101:9463; Rodriguez-Viejo et al., 1997, Appl. Phys. Lett. 70: 2132-2134;Peng et al., 1997, J. Am. Chem. Soc. 119: 7019-7029; 1996, Phys. ReviewB. 53: 16338-16346; the disclosures of which are hereby incorporated byreference). The core nanocrystal with the capping layer may include 0 toabout 5.3 monolayers of the capping semiconductor material, preferablyit includes less than about 1 monolayer of the capping semiconductormaterial. As known to those skilled in the art, the size of the core ofthe semiconductor nanocrystal correlates with the spectral range ofemission. Table 1 is an illustrative example for CdSe. TABLE 1 ColorNanocrystal Size Range (nm) Peak Emission Range (nm) Blue 2.1 to 2.9 436to 500 Green 2.9 to 4.7 500 to 575 Yellow 4.7 to 5.0 575 to 592 Orange5.0 to 6.1 592 to 620 Red  6.1 to 10.2 620 to 650

[0035] In a preferred embodiment, the semiconductor nanocrystals areproduced using a continuous flow process and system (see, U.S. Pat. No.6,179,912), and may have a particle size that varies by less than +/−10%in the average particle size (as measured by diameter) in the range ofapproximately 1 nanometer (nm) to approximately 20 nm. Semiconductornanocrystals useful in the practice of various embodiments of thisinvention may also be characterized in that the dimensions of thenanocrystals are comparable or smaller than their bulk exciton diameterso that they exhibit size dependent optoelectronic properties.

[0036] By the term “doped metal oxide nanocrystals” is meant, forpurposes of the specification and claims to refer to nanocrystalscomprised of: a metal oxide, and a dopant comprised of one or more rareearth elements. For example, metal oxides include, but are not limitedto yttrium oxide (Y₂O₃), zirconium oxide (ZrO₂), zinc oxide (ZnO),copper oxide (CuO or Cu₂O), gadolinium oxide (Gd₂O₃), praseodymium oxide(Pr₂O₃), lanthanum oxide (La₂O₃), and alloys thereof. Doped metal oxidenanocrystals with rare earth elements may include but are not limited tooxides of elements selected from the Lanthanide series such as europium(Eu), cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Tb),gadolinium (Gd), holmium (Ho), thulium (Tm), and alloys containing theseelememts. As known to those skilled in the art, depending on the dopant,an energized doped metal oxide nanocrystal is capable of emitting lightof a particular color. Thus, the nature of the rare earth or rare earthsare selected in consequence to the color sought to be imparted (emitted)by a doped metal oxide nanocrystal used to label a microsphere accordingto the present invention. A given rare earth or rare earth metalcombination in a doped metal oxide has a given color. By adjusting thenature of the dopant and or the concentration of the dopant, the dopedmetal oxide nanocrystals may emit (with a narrow emission peak) a colorover an entire range of colors. For example, the emission color andbrightness (e.g., intensity) of a doped metal oxide nanocrystalcomprising Y₂O₃:Eu may depend on the concentration of the Eu dopant;e.g., emission color may shift from yellow to red with increasing Euconcentration. For purposes of illustration only, representative colorswhich may be provided by various dopants in Y₂O₃ are listed in Table 2.TABLE 2 Fluorescent Color Dopant Blue Thulium Blue Cerium Yellow-greenTerbium Green Holmium Green Erbium Red Europium Reddish Samarium OrangeNeodymium Yellow Dysprosium White Praseodymium Orange-yellow Europium +terbium Orange-red Europium + samarium

[0037] Methods for making doped metal oxide nanocrystals are known toinclude, but are not limited to a sol-gel process (see, e.g., U.S. Pat.No. 5,637,258), and an organometallic reaction. As will be apparent toone skilled in the art, the dopant (e.g., one or more rare earthelements) are incorporated into the doped metal oxide nanocrystal in asufficient amount to permit the doped metal oxide nanocrystal to be putto practical use in fluorescence detection. Without enough dopant, thedoped metal oxide nanocrystal would fail to emit sufficient detectablefluorescence, too much dopant which would cause reduced fluorescence dueto concentration quenching. In a preferred embodiment, the amount ofdopant in a doped metal oxide nanocrystal is a molar amount in the dopedmetal oxide nanocrystal selected in the range of from about 0.1% toabout 25%. Doped metal oxide nanocrystals may be excited with a singleexcitation light source resulting in a detectable fluorescence emissionof high quantum yield (e.g., a single nanocrystal having at afluorescence intensity that may be a log or more greater than that amolecule of a conventional fluorescent dye) and with a discretefluorescence peak. Typically, doped metal oxide nanocrystals have asubstantially uniform size of less than 200 Angstroms, and preferablyhave a substantially uniform size in the range of sizes of from about 1nm to about 5 nm. In one embodiment, the doped metal oxide nanocrystalsare comprised of metal oxides doped with one or more rare earthelements, wherein the dopant comprising the rare earth element iscapable of being excited (e.g., with ultraviolet light) to produce anarrow spectrum of fluorescence emission. In another embodiment, thedoped metal oxide has both fluorescent properties (when excited with anexcitation light source) and magnetic properties; thus, a polymericmicrosphere (which is substantially nonmagnetic) embedded or labeledwith a plurality of fluorescent nanocrystals (comprising doped metaloxide nanocrystals which are magnetic material) may form fluorescentmicrospheres which are magnetic.

[0038] By the term “functionalized fluorescent nanocrystals” is meant,for purposes of the specification and claims to refer to fluorescentnanocrystals which are coated. The coating may include but is notlimited to cations, ligands, molecules with conjugated moieties as wellas lyophilic and bonding moieties, cross linking molecules, andmolecular probes. An example of such a material coating is animidazole-containing compound and alkyl phosphine containing compound.

[0039] Functionalized fluorescent nanocrystals according to the presentinvention are soluble in aqueous solutions and other fluids dependingupon the ligands comprising the material coating. For example, they maybe soluble in water, water and isopropyl alcohol mixtures or liquidswith surface tensions below about 80 dynes/cm, and preferably in therange from about 30-73 dynes/cm. The solvent or solvent mixture used tosolublize or suspend the functionalized nanocrystals may have a surfaceenergy which is about the same as the surface energy of the particularcoating material of the functionalized nanocrystal. The surface energyof coating will vary with the molecular properties and amount of theligands in the coating material; preferably the moiety of the coatingmaterial is compatible, soluble and chemically stable, with the fluidsit is contacted with. Chemically stable moieties of the coating materialmaintain the fluorescent emission intensity over time as required by theapplication of the functionalized fluorescent nanocrystals. Thefunctionalized nanocrystals (FNC) of the present invention were solublein water, mixtures of water and glycerol (50%), water and ethanol (10%),water and methanol (50%; ˜35 dynes/cm), waer and DMSO (50%), water andpolyethylene glycol 200 (50%), and water and isopropyl alcohol (50%).The FNC were also soluble in 100% of glycerol, isopropyl alcoholparticularly after adding other solvents to isopropyl alcohol. Thefunctionalized fluorescent may operably bond molecular probes and targetmolecules, have increased fluorescence intensity when excited by asuitable excitation source; and may further demonstrate chemicalstability in a pH range of from about pH 6.0 to about 10.5. Preferredfunctionalized, fluorescent nanocrystals may be produced, and used inthe method and system as according to present invention, to theexclusion of functionalized, fluorescent nanocrystals other than thepreferred functionalized, fluorescent nanocrystals.

[0040] In a preferred embodiment to form the functionalized, fluorescentnanocrystals according to the present invention, a core nanocrystal maybe coated by the co-precipitation of a compound comprising a metalcation (e.g., for forming a semiconductor material, preferably, with ahigh bad gap energy, as known in the art; “shell”) operably bound to animidazole-containing compound and to an alkyl phosphine-containingcompound, wherein the coat is uniformly deposited over the outer surfaceof the nanocrystal core. This is both functionally and fundamentallydifferent than using zinc-histidine as nucleation centers for growingcore nanocrystals (see, Kho et al., 2000, Biochem. Biophys. Res. Commun.272: 29-35). As an example of this preferred embodiment, a Group II-VIsemiconductor core may be capped with a Group II-VI semiconductor shell(e.g., a ZnS or CdSe core may be coated with a shell comprised of YZwherein Y is Cd or Zn, and Z is S, or Se) and further coated with animidazole-containing compound cross-linked with an alkylphosphine-containing compound. Preferably both the semiconductor shelland imidazole-containing compound cross linked with the alkylphosphine-containing compound coating passivate the outer surface of thecore nanocrystal and result in enhanced fluorescence.

[0041] In another embodiment, a core/shell nanocrystal (e.g., CdXcore/YZ shell) produced using methods standard in the art is coated witha metal cation (preferably capable of forming a semiconductor material,like ZnS, with a high band gap energy) operably bound to animidazole-containing compound cross-linked with an alkylphosphine-containing compound. The coating is uniformly deposited overthe outer surface of the core/shell nanocrystal.

[0042] In another embodiment, a fluorescent nanocrystal may be coatedwith a material that has an imidazole-containing compound cross-linkedwith alkyl phosphine-containing compound that produces thefunctionalized fluorescent nanocrystals according to the presentinvention. The functionalized fluorescent nanocrystal composition mayluminescence with an efficiency of greater than about 10-fold of that ofnon coated fluorescent nanocrystals when irradiated with light or othersource of exitation.

[0043] In yet another embodiment, a fluorescent nanocrystal may becoated with a coating comprising an imidazole-containing compound andalkyl phosphine-containing compound.

[0044] In another embodiment, the functionalized, fluorescentnanocrystals according to the present invention further compriseschemical or physical cross-linking of the coating material comprisingimidazole-containing compound and alkyl phosphine-containing compound topromote further stabilization of the coat of the functionalized,fluorescent nanocrystal. Chemical cross-linking can be achieved by usingmethods and reagents known in the art which may include, but are notlimited to, formaldehyde, glutaraldehyde, acrolein,1,6-hexane-bis-vinylsulfone, putrescine, alkyl diamines, and otherorganic triamines or polyamines. Physical cross-linking and/or curingcan also be achieved by using methods known in the art which mayinclude, but are not limited to, ultraviolet irradiation, microwavetreatment, heat treatment, and radiation.

[0045] Excitation sources suitable for characterizing the functionalizedfluorescent nanocrystals of this invention include but are not limitedto polychromatic ultraviolet and visible lamps, substantiallymonochromatic sources light, polarized light, beta emitters includingbut not limited to ³³P, ¹²⁵I, and ³H. Sources of light may include low,medium, and high pressure lamps as well as lasers. Electric current andelectron bombardment of the nanocrystals may also me used forexcitation. Suitable detectors may include but are not limited to visualdetection, photodiodes, photomultipliers, heat detectors and chargecoupled device detectors (CCDs); detectors may also include the use ofpolarizing filters.

[0046] The functionalized fluorescent nanocrystal prepared by themethods and materials of embodiments of the present invention may becharacterized by luminescent efficiency or quanum yield. For example,fluorescent nanocrystals that have been coated with an imidazolecontaining or ligand with delocalized electrons operably bonded to thenanocrystal may be excited with a light source and the number of photonsfrom the fluorescence measured to characterize the Quantum Yield of thecomplex. Alternatively, the coated fluorescent nanocrystals may beconnected to anodes and cathodes as know to those skilled in the art,and luminescent output based on injected charge used to characterize theluminescent efficiency of the functionalized fluorescent nanocrystal.Adjustment to the amount and type of imidazole containing ligand,phosphine cross linker, cross linker, or other parameters may be madeand used to change the luminescent properties of the coated crystals.

[0047] An effective amount of a coating is an amount of coating wherethe steric interactions of the coating molecules (determined by size,intermolecular bonding, cross linking, and operable bonding to targetmolecules) and their electronic interaction with the fluorescentnanocrystal are such that the functionalized fluorescent nanocrystal hasa quantum yield in excess of 10% and preferably in excess of 50% whenexcited with light.

[0048] The present invention provides compositions comprisingfunctionalized, fluorescent nanocrystals which can be used in a varietyof types of fluorescence-based detection systems. Examples include, butare not limited to, building three dimensional dendrimers which functionto generate and significantly amplify a detectable signal (therebyconsiderably improving the sensitivity of a non-isotopic detectionsystem; see, e.g., U.S. Pat. No. 6,261,779, the disclosure of which isherein incorporated by reference). Another example is to use thefunctionalized fluorescent nanocrystals of the present invention tolabel nucleobases and provide fluorescence-labeled nucleobases fornucleic acid strand synthesis or nucleic acid sequence determination(see, e.g., U.S. Pat. No. 6,221,602, the disclosure of which is hereinincorporated by reference). Another example is to use the functionalizedfluorescent nanocrystals of the present invention to make fluorescentmicrospheres (e.g., beads) by either embedding microspheres with and/orto operably bind microspheres to functionalized fluorescentnanocrystals. Another example is to use the functionalized fluorescentnanocrystals of the present invention in fluorescent ink compositionssuitable for printing on substrates. In this example, the functionalizedfluorescent nanocrystal ink composition is applied to a substrate in anidentifiable code pattern and is then excited with a suitable forpurposes of identification, verification, security, or ornamentation.

[0049] As will be apparent to one skilled in the art, thefunctionalized, fluorescent nanocrystals according to the presentinvention may be used in a detection system that may include, but is notlimited to, one or more of: an affinity assay (e.g., immunoassay such asan ELISA), fluorescent staining (e.g., immunofluorescent staining on aglass slide, fluorescent in situ hybridization, and the like), flowcytometry, cell imaging-based detection assays (e.g., cell-based ELISAor “cELISA”, image cytometry, cells grown in standard high densitymicroarrays), microarray-based detection assays (e.g., olignucleotidescanning arrays, combinational DNA arrays, microchips containing arraysof nucleic acid molecules or protein molecules, multi-channel microchipelectrophoresis, and the like), microfluidics-based detection assays(e.g., “lab-on-a-chip” systems as known in the art), fluorescence-basedbiosensors (see, e.g., Trends in Biotech. 16: 135-140, 1999), nucleicacid sequencing, nucleic acid hybridization, nucleic acid synthesis oramplification, manufacturing of light emitting diodes, identificationverification (e.g., identification card or bank card), fluorescentbead-based detection assays, molecular sorting (e.g., cell sorting byflow cytometry), and the like.

[0050] This example illustrates embodiments of a process of making thefunctionalized, fluorescent nanocrystals according to the presentinvention. For this and subsequent examples, semiconductor nanocrystalscomprising core nanocrystals were produced using a continuous flowprocess as described in the U.S. Pat. No. 6,179,912. The followingparameters were used to produce nanocrystals of cadmium selenide (CdSe):10 g TOPO; 18.9 ul of Me₂Cd (dimethyl cadmium; e.g., 2.63×10⁻⁴ moles ofCd); 198.9 ul of TOPSe (1M solution of Se in TOP; e.g., 1.989×10⁻⁴ molesof Se); 4.5 ml of TOP; nucleation temperature (Tn) of 300 C; growthtemperature (Tg) of 280° C.; and flow rate of 0.1 ml/min. The resultingCdSe nanocrystals displayed fluorescence at a wavelength of 578 nm, withan excitation wavelength of 410 nm, and a narrow bandwidth at halfheight of about 29 nm.

[0051] The process of making functionalized, fluorescent nanocrystalsmay comprise contacting fluorescent nanocrystals, for example CdSe, Zn Sor (CdSe)ZnS with a solution comprising a ligand or animidazole-containing compound and then with a solution comprisinganother ligand such as an alkyl phosphine-containing compound. Whenmetal ions like Cd⁺² or Zn⁺² are present in solution or on thefluorescent nanocrystal, the ligand or the imidazole-containing compoundoperably binds to the metal cation and the alkyl phosphine-containingcompound operably binds to the counterpart non metal element (e.g., S,Se, or the like) in producing a coat over the fluorescent nanocrystalsin forming functionalized, fluorescent nanocrystals.

[0052] As previously described, the fluorescent nanocrystals which arecoated by the process may comprise core semiconductor nanocrystals,core/shell semiconductor nanocrystals, doped metal oxide nanocrystals,or a combination thereof. With respect to metal cations, ligands orother imidazole-containing compounds have been reported to operably bindmetal ions which may include, but are not limited to one or more of,Zn²⁺, Cu²⁺, Fe²⁺, Hg⁺², Ni²⁺, Cd²⁺, Co²⁺, and the like. With respect tonon metal counter part anions, alkyl phosphine-containing compounds havebeen reported to operably bind to non metal elements which may include,but are not limited to one or more of, O, S, Se, Te, Po and the like.

[0053] For example, core fluorescent nanocrystals are prepared andfirstly coated by an inorganic layer of ZnS according to methods knownin the art to form core/shell type nanocrystals, and then the core/shelltype nanocrystals are coated by a second coating solution comprising aligand or an imidazole-containing compound and then with a solutioncomprising an alkyl phosphine-containing compound. The ligand orimidazole-containing compound operably binds to the metal cation ifpresent (e.g., Cd⁺², Zn⁺² or the like) and the alkylphosphine-containing compound operably binds to the counterpart nonmetal element (e.g., S, Se, or the like) in producing a coat over thefluorescent nanocrystals in forming functionalized, fluorescentnanocrystals. As a general guideline, the core/shell nanocrystalscoating process may comprise inclusion of components comprising, permilligram of fluorescent nanocrystals: ligands or animidazole-containing compound in an amount ranging from about 0.25 mmoleto about 2.5 mmole; alkyl phosphine-containing cross-linker in an amountranging from about 0.25 mmole to about 2.5 mmole; and a polyamine in anamount ranging from about 0 mmole to about 2.5 mmole. As apparent to oneskilled in the art, the amount of each individual component may varydepending on the particular ligand or imidazole-containing compoundused, the alkyl phosphine-containing compound used, the nature (e.g.,chemical composition) of fluorescent nanocrystals to be functionalized,the nature of the surface of the fluorescent nanocrystals to be coated,the ratio of other components in the coating process, and the pH of thebuffer system used in the coating process.

[0054] In one embodiment of the coating process may be monitored by theemission of the fluorescent nanocrystals during the coating process.Reaction conditions such as time, temperature and concentration ofreagents may be adjusted according to the emission of excitedfunctionalized fluorescent nanocrystals during the process. A thresholdemission intensity may be chosen to indicate a desired state of thecoating process. For example, the coating process may continue until theemission from the fluorescent nanocrystals during the process is atleast the intensity of the threshold value.

[0055] For example, a 30 mM carnosine (imidazole containing compound)solution in a 1 M CAPSO buffer(3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, sodium salt, pH9.6) was prepared. Other suitable buffers known in the art which providebuffering in a range of from about pH 8.0 to about pH 11, may be used inplace of the CAPSO buffer (e.g., a sodium carbonate buffer, TAPS buffer(N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid), and thelike). To 10 ml of the carnosine solution was added 0.5 to 3 mg of CdSenanocrystals (core crystals) suspended in a minimal volume (e.g., fromabout 60 ul to about 200 ul) of organic solvent (e.g., chloroform orpyridine). After mixing, and incubation for about 1 hr at roomtemperature, the organic phase was discarded Then to the aqueous phasewas added 1.2 ml of 60 mM THPP(beta-[Tris(hydroxymethyl)phosphino]propioninc acid, alkylphosphine-containing cross-linker)). The temperature of the reactionsmay be modified as would be known to those skilled in the art to affectthe coating process. After one hour of gentle mixing, 100 ul of 1Mputrescine (polyamine) was added and mixed for additional hour. Thecycle of the addition of THPP and putrescine was repeated three to fourtimes. The final solution was treated with formaldehyde at 100 mM finalconcentration for about 1 hour period. The functionalized, fluorescentnanocrystals were then purified. Suitable methods of purificationinclude but are not limited to size exclusion chromatography, dialysis,centrifugation, and a combination these methods. For example thesolution comprising functionalized, fluorescent nanocrystals wasdialyzed against a suitable buffer such like PBS (phosphate bufferedsaline) using 3000 KD MCO dialysis membranes.

[0056] The process for making the functionalized, fluorescentnanocrystals was repeated and the relative amounts of each componentwere varied. The resultant functionalized, fluorescent nanocrystals werecharacterized by: stability in aqueous solutions in a pH range of about6 to about 10, with optimal stability in the range of from about pH 7 toabout pH 9; available reactive functionalities on the surface of thefunctionalized, fluorescent nanocrystals (in this case, carboxyl groups)to which molecular probe may be operably bound; and fluorescenceintensity. There are two measures of stability which may be used; thefirst, is decay in fluorescence intensity over time to a threshold ofthe initial value etc. Most stable crystals decay 1% in 24 hours, leaststable decay 25% in 24 hours. The second measure of stability is thephysical stability were there is no change in solubility, aggregation,cloudiness, or phase separation, stability toward repeatedcentrifugation and filtration, and toward dialysis and electrophoresis.From these formulation studies, a preferred ratio of components thatshowed optimal properties of fluorescence and stability (in an aqueousenvironment and at a wide pH range) comprises: 1 to 2 mg of core/shellnanocrystals (e.g., CdSe/ZnS); 0.35 mmole carnosine; 0.15 mmole THPP;0.15 mmole putrescine, and 1 mmole formaldehyde.

[0057] These functionalized fluorescent nanocrystals showed unexpectedenhancement of fluorescence intensity comprising at least 50 fold to asmuch as about 1100 fold or more (see, e.g., FIG. 2 and inset of FIG. 2),when compared to the fluorescence intensity of functionalizedfluorescent nanocrystals known in the art (e.g., CdX/YZ fluorescentnanocrystals in organic solvent or CdX/YZ fluorescent nanocrystalscapped with mercapto-based compound) (see, e.g., FIG. 2). Thecharacteristic spectral emission of the coated fluorescent nanocrystalis enhanced by the coating material. The comparison of fluorescenceintensity was made by comparison to an equivalent amount of fluorescentnanocrystals made using core/shell nanocrystals from the samepreparation; excitation with the same excitation light source (e.g., 410nm); and detection using the same detection system.

[0058] In this example, provided is another embodiment of process ofmaking functionalized, fluorescent nanocrystals by coating fluorescentnanocrystals with a coating comprising histidine as a ligand orimidazole-containing compound. As a general guideline, the core/shellnanocrystals coating process may comprise inclusion of componentscomprising, per milligram of fluorescent nanocrystals: a ligand orimidazole-containing compound in an amount ranging from about 0.25 mmoleto about 2.5 mmole; alkyl phosphine-containing cross-linker in an amountranging from about 0.25 mmole to about 2.5 mmole; and a polyamine in anamount ranging from about 0 mmole to about 2.5 mmole. As apparent to oneskilled in the art, the amount of each individual component may varydepending on the particular ligand or imidazole-containing compoundused, the alkyl phosphine-containing compound used, the nature (e.g.,chemical composition) of fluorescent nanocrystals to be functionalized,the nature of the surface of the fluorescent nanocrystals to be coated,the ratio of other components in the coating process, and the pH of thebuffer system used in the coating process.

[0059] For example, prepared was a 30 mM histidine (imidazole containingcompound) solution in a 1 M CAPSO buffer(3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, sodium salt, pH9.6). Other suitable buffers known in the art may be used. To 10 ml ofthe histidine solution was added 0.5 to 3 mg of CdSe nanocrystals (corecrystals) suspended in a minimal volume (e.g., from about 60 ul to about200 ul) of organic solvent (e.g., chloroform or pyridine). After mixing,and incubation for about 1 hr at room temperature, the organic phase wasdiscarded. Then to the mixture was added 1.2 ml of 60 mM THPP(beta-[Tris(hydroxymethyl)phosphino]propioninc acid, alkylphosphine-containing cross-linker). After one hour of gentle mixing, 100ul of 1M putrescine (polyamine) was added and mixed for additional hour.The cycle of the addition of THPP and putrescine was repeated three tofour times. The final solution was treated with formaldehyde at 100 mMfinal concentration for about 1 hour period. The functionalized,fluorescent nanocrystals were then purified as previously described.

[0060] This process for making the functionalized, fluorescentnanocrystals was repeated, whereby the relative amounts of eachcomponent were varied. The resultant functionalized, fluorescentnanocrystals were characterized by: stability in aqueous solutions ofthe general pH range of about 6 to about 10, with optimal stability inthe range of from about pH 7 to about pH 9; availability of reactivefunctionalities on the surface of the functionalized, fluorescentnanocrystals (in this case, carboxyl groups) to which molecular probemay be operably bound; and fluorescence intensity. From theseformulation studies, a preferred ratio of components that showed optimalproperties of fluorescence and stability (in an aqueous environment andat a wide pH range) comprises: 1 to 2 mg of core/shell nanocrystals(e.g., CdSe/ZnS); 0.35 mmole histidine; 0.15 mmole THPP; 0.15 mmoleputrescine, and 1 mmole formaldehyde.

[0061] These functionalized fluorescent nanocrystals showed unexpectedenhancement of fluorescence intensity, about 50 fold to as much as about1100 fold or more, when compared to fluorescence intensity offunctionalized fluorescent nanocrystals known in the art (e.g., CdX/YZfluorescent nanocrystals in organic solvent or CdX/YZ fluorescentnanocrystals capped with mercapto-based compound). The comparison offluorescence intensity was made as previously described.

What is claimed:
 1. A functionalized fluorescent nanocrystal comprisedof: a fluorescent nanocrystal; a coating material, said coating materialbeing histidine-based
 2. The functionalized fluorescent nanocrystal ofclaim 1, wherein said fluorescent nanocrystal emits light with a quantumyield of greater than about 10%
 3. The functionalized fluorescentnanocrystal of claim 1, wherein said fluorescent nanocrystal emits lightwith a quantum yield of greater than about 30%.
 4. The functionalizedfluorescent nanocrystal of claim 1, wherein said fluorescent nanocrystalemits light with a quantum yield of greater than about 50%.
 5. Thefunctionalized fluorescent nanocrystal of claim 1, wherein saidfluorescent nanocrystal emits light with a quantum yield of greater thanabout 70%.
 6. The functionalized fluorescent nanocrystal of claim 1,wherein said coating material includes carnosine.
 7. The functionalizedfluorescent nanocrystal of claim 1, wherein the fluorescent nanocrystalis soluble in a fluid with a surface energy of greater than 35 dynes/cm.8. The functionalized fluorescent nanocrystal of claim 1, wherein saidfluorescent nanocystals are soluble in an aqueous solution.
 9. Thefunctionalized functionalized fluorescent nanocrystal of claim 1,wherein said probe includes a polynucleotide of known sequence attachedto said nanocrystal via an amino terminal portion of saidpolynucleotide.
 10. The functionalized fluorescent nanocrystal of claim1, wherein said probe includes a polynucleotide of known sequenceattached to said nanocrystal via a carboxy terminal portion of saidpolynucleotide.
 11. The functionalized fluorescent nanocrystalcomposition of claim 1 wherein said fluorescent nanocyrstal is cadmiumselenide; and said coating material comprises zinc sulfide operablybonded to cross linked histidine and(beta-(tris(hydroxymethyl)phosphino)propionic acid)).
 12. Thefunctionalized fluorescent nanocrystal composition of claim 1 whereinsaid fluorescent nanocrystal is cadmium selenide; and said coatingmaterial comprises zinc sulfide operably bonded to cross linkedcarnosine and (beta-(tris(hydroxymethyl)phosphino)propionic acid)). 13.The functionalized fluorescent nanocrystal composition of claim 1wherein said molecular probe is chosen from the group consisting ofavidin, ConA, lectin, and IgG.
 14. The functionalized fluorescentnanocrystal composition of claim 13 wherein said molecular probe isavidin.
 15. The fluorescent nanocrystal of claim 1 wherein saidnanocrystal is a doped metal oxide nanocrysal.
 16. Fluorescentnanocrystal of claim 1 wherein said doped metal oxide nanocrystal ismagnetized when irradiated with light.
 17. The functionalizedfluorescent nanocrystal of claim 1 wherein said fluorescent nanocrystalshave a characteristic spectral emission intensity which is enhanced bythe coating material.
 18. The composition of claim 1 wherein thefluorescent nanocrystal comprises a core material and a capping layermaterial.
 19. The composition of claim 18 wherein said core material isCdSe and said capping layer material is ZnS.
 20. The composition ofclaim 17 wherein said emission provides information about a biologicalstate or event.
 21. A method for detecting a target species, the methodcomprising: exciting a complex formed by operably bonding a fluorescentnanocrystal to a target molecule to form a complex wherein saidfluorescent nanocrystal includes a imidazole-based coating material; anddetecting the light emitted from said complex.
 22. The method of claim21 wherein the detecting further comprises detecting the intensity oflight emitted from said complex in the perpendicular and paralleldirections.
 23. A method for separating materials comprising:introducing doped metal oxide fluorescent nanocrystal soluble in fluidscoated with the composition of claim 1 into a system; exciting saidfluorescent nanocrystal to have a magnetic moment; and utilizing amagnetic field for separating the doped metal oxide fluorescentnanocrystals with the induced magnetic moment.
 24. The method of claim23 wherein said doped metal oxide fluorescent nanocrystals furthercomprise a molecule operably to said doped metal oxide fluorescentnanocrystal.
 25. The method of claim 24 wherein said molecule is chosenfrom the group consisting of target molecules and molecular probes. 26.A functionalized fluorescent nanocrystal composition comprising afluorescent nanocrystal, a coating material, and a molecular probewherein said fluorescent nanocrystals have a characteristic spectralemission which is enhanced by the coating material.
 27. The compositionof claim 26 wherein the coating material is a histidine-basedcomposition.
 28. The composition of claim 26 wherein the coatingmaterial is a carnosine-based composition.
 29. The composition of claim26 wherein the fluorescent nanocrystal comprises a core material and acapping layer material.
 30. The composition of claim 29 wherein saidcore material is CdSe and said over-coating material is ZnSe.
 31. Thecomposition of claim 26 wherein said emission provides information abouta biological state or event.
 32. A coated nanocrystal capable of lightemission comprising a core comprising: a first semiconductor material,an capping layer deposited on the core comprising a second semiconductormaterial, a coating material deposited on said capping layer including ahistidine-based composition coating material wherein said coatednanocrystal has a quantum yield of greater than 50% when irradiated withlight.
 33. The coated nanocrystal of claim 32 wherein the secondsemiconductor material is ZnS.
 34. The coated nanocrystal of claim 32wherein the capping layer is from about one to about two monolayers ofthe second semiconductor material.
 34. The coated nanocrystal of claim32 wherein the second semiconductor material is ZnSe.
 35. The coatednanocrystal of claim 32 wherein the nanocrystal further comprises anorganic layer on the nanocrystal coating layer.
 36. The coatednanocrystal of claim 35 wherein the organic layer comprises a moietyselected to provide compatability with a suspension medium.
 37. Thecoated nanocrystal of claim 35 wherein the organic layer comprises amoiety selected to exhibit affinity for the outer surface of thenanocrystal.
 38. The coated nanocrystal of claim 37 wherein the organiclayer comprises a short-chain polymer terminating in a moiety havingaffinity for a suspending medium.
 39. The coated nanocrystal of claim 32wherein the first semiconductor material is selected from the groupconsisting of CdS, CdSe, CdTe, and mixtures thereof.
 40. The coatednanocrystal of claim 39, wherein the second semiconductor material has ahigher band gap than the first semiconductor material.
 41. The coatednanocrystal of claim 32 wherein the second semiconductor material isselected from the group consisting of ZnS, ZnSe, CdS, CdSe, and mixturesthereof.
 42. The coated nanocrystal of claim 32 wherein the firstsemiconductor material is CdSe and the second semiconductor material isZnS.
 43. The coated nanocrystal of claim 42, wherein the capping layercomprises greater than about 0 to about 5.3 monolayers of the secondsemiconductor material.
 44. The coated nanocrystal of claim 43, whereinthe capping layer comprises less than about one monolayer of the secondsemiconductor material.
 45. The coated nanocrystal of claim 44, whereinthe second semiconductor material is ZnS or ZnSe.
 46. The coatednanocrystal of claim 43, wherein the capping layer comprises in therange of about one to about two monolayers of the second semiconductormaterial.
 47. The coated nanocrystal of claim 43, wherein the secondsemiconductor material is ZnS or ZnSe.
 48. The coated nanocrystal ofclaim 32, wherein said coated nanocrystal is a member of a substantiallymonodisperse particle population.